Project summary: Increased central vascular stiffness in aging is an independent predictor of major adverse cardiovascular events and the leading cause of morbidity and mortality in the elderly. While mechanisms of vascular stiffening are emerging, targeted therapies are not yet available. Vascular stiffening and its sequelae remain clinically challenging to manage and currently, care relies on lifestyle modifications and pharmacologic agents targeting essential hypertension despite the distinct etiology and cellular molecular mechanisms, leading to syncope and falls in the elderly. and with the shift of population demographics towards the elderly, the clinical, societal, and financial burden of vascular stiffening has grown. Therefore, there is an urgent need to identify and characterize potential targets for clinical management of vascular stiffening and mitigate its sequelae. Using an unbiased proteomic screen, we have identified lysyl oxidase like 2 (LOXL2) as a novel protein that mediates vascular stiffening. Our hypothesis, strongly supported by preliminary data, is that LOXL2 regulates matrix composition, deposition, and architecture, modulates vascular smooth muscle cell (VSMC) micromechanics and contractility, and contributes to aging associated increase in vascular stiffness in mice. In this grant, we propose to examine the mechanisms of LOXL2-mediated vascular stiffening in aging in a hierarchical fashion from cells to mouse models of aging. We have generated novel reagents, mouse models, and assembled a strong, multidisciplinary research team to successfully execute the proposed work. In Aim 1, we will determine the mechanism of LOXL2 regulation by nitric oxide and proteolytic processing by Factor Xa in VSMCs. These aspects of LOXL2 regulation in cells and vasculature are not yet known. In Aim 2, we postulate that the scavenger receptor cysteine rich (SRCR) domains and amine oxidase (LOX) domains present in LOXL2 have distinct functions that contribute uniquely to vascular stiffening. We will 1) define the mechanisms of ECM remodeling by LOXL2 with specific emphasis on its putative scaffolding function and 2) determine how LOXL2 modulates VSMC micromechanics, cytoskeletal dynamics, and behavior/function. In Aim 3, we will determine if targeting LOXL2 mitigates age-associated vascular stiffening in mouse models. Using a combination of genetic (tamoxifen inducible global, VSMC-specific, and EC-specific LOXL2-/- mice), and pharmacologic (PAT-1251) approaches to target LOXL2, we will identify 1) the cellular source of LOXL2 in the aging vessel, 2) investigate if LOXL2 targeting can halt or reverse vascular stiffening in aging, and 3) identify whether VSMC stiffening or ECM deposition is the primary mechanism by which LOXL2 acts in the aging vessel. In sum, these studies will advance our knowledge of LOXL2 protein biochemistry and biology, establish the mechanisms by which LOXL2 is regulated in the vasculature, determine the mechanisms by which LOXL2 drives age-associated vascular stiffening, and lay the scientific foundation for the discovery and development of novel and effective therapies for the clinical management of vascular stiffening.